Renovation and Repopulation of Decellularized Tissues and Cadaveric Organs by Stem Cells

ABSTRACT

A method of manufacturing a tissue matrix for implantation into a patient is disclosed. The method sets forth collecting embryonic stem cells from a placenta which has been treated to remove residual cord blood and seeding the collected stem cells onto or into a tissue matrix. The seeded tissue matrix is then implanted on or into a patient. The seeded tissue matrix made by the method of the present invention is also disclosed.

BENEFIT OF PRIOR PROVISIONAL APPLICATION

This utility patent application claims the benefit of co-pending priorU.S. Provisional Patent Application Ser. No. 60/268,560, filed Feb. 14,2001, entitled “Renovation and Repopulation of Decellularized CadavericOrgans by Stem Cells” having the same named applicant as inventor,namely Robert J. Hariri.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is generally in the field of tissue engineering,and more specifically is a means for obtaining stem cells for seedingonto scaffolding for regeneration or repair of tissue, bone and otherorgans.

2. Description of the Background Art

The scarcity of human donor organs for transplantation is a growingproblem. Despite aggressive public awareness campaigns the numbers ofqualified organ donors has changed little in the last 20 years while thedemand has grown at a rapid pace. In addition, allogeneic organtransplantation is still associated with a high frequency ofcomplications due to immune rejection. Attempts to address this crisishave included development of ex vivo and implantable synthetic organsupport devices such as pump devices for cardiac support and use oforgans from other species (xenotransplantation). Xenotransplantation hasbeen refined to include development of chimeric donor animals yet isstill unperfected and subject to possible consequences such as thetransmission of zoonoses.

Human stem cells are totipotential or pluripotential precursor cellscapable of generating a variety of mature human cell lineages. Thisability serves as the basis for the cellular differentiation andspecialization necessary for organ and tissue development. Recentsuccess at transplanting such stem cells have provided new clinicaltools to reconstitute and/or supplement the bone marrow aftermyeloablation due to disease, exposure to toxic chemical or radiation.Further evidence exists which demonstrates that stem cells can beemployed to repopulate many, if not all, tissues and restore physiologicand anatomic functionality. Evidence to date indicates that multipotentor pluripotent stem cells are directed to differentiate into specificmature cell lineages based on the physical and biochemical environmentthat they are delivered. There is also evidence that these cells canmigrate from normal to abnormal or defective tissues and repopulatethose areas in a very focused and specific manner.

The application of stem cells in tissue engineering, gene therapy andcell therapeutics is also advancing rapidly.

Many different types of mammalian stem cells have been characterized.For example, embryonic stem cells, embryonic germ cells, adult stemcells or other committed stem cells or progenitor cells are known.Certain stem cells have not only been isolated and characterized buthave also been cultured under conditions to allow differentiation to alimited extent.

Despite considerable advances made in controlling differentiation ofstem cells into mature cells and tissues, actual development of thecomplex architecture of solid organs has not been accomplished. Tissueengineering has therefore directed attention at growing componenttissues and then assembling those components into useful structures.

It is therefore an object of the present invention to provide methods toremove the cellular content of tissues while preserving theextracellular matrix architecture coupled with advanced understanding ofstem cells, which can be seeded with cells to yield whole organs withthe anatomic and physiologic features of native organs.

SUMMARY OF THE INVENTION

Cadaveric solid organs are processed to remove all living cellularcomponents yet preserve the underlying extracellular matrix scaffold inpreparation for the implantation of allogeneic stem cells whichrepopulate the three-dimensional organ scaffold and restore normalanatomic and physiologic function for the purpose of organtransplantation. In a preferred embodiment, the cells are obtained fromplacental tissue. The method for the processing of cadaveric solidorgans selectively depletes the cellular components of the organ whilepreserving the native biochemical and 3-dimensional architecture of saidorgan. The resulting organ scaffold or ‘template’ is then implanted withgenotype-specific, living multi-potent stem cells which are delivered byintraparenchymal injection or through the organ's vascular tree.Delivery of these cells is made in a manner that promotes thedifferentiation and proliferation of the normal mature cell types of theorgan, the distribution and ultimate cell to cell assembly being guidedby the extracellular matrix scaffold. The repopulation of the organscaffold takes place under environmentally-controlled culture conditionsproviding immersion in and perfusion with tissue culture mediaformulated to deliver the optimal nutrient and metabolic levelsnecessary to sustain the organ, while simulating those biomechanicalforces found in vivo. The system monitors the physiologic and metabolicstate of the organ during repopulation and renovation and adjustsconditions as needed to maintain the optimal steady state. Repopulatedorgans can then be maintained under these support conditions until suchtime that they can be tested and transplanted.

The present invention relates to methods of manufacturing a tissue ororgan in vivo. The methods of the invention encompass usingembryonic-like stem cells obtained from a placenta which has beentreated to remove residual cord blood to seed a matrix and culturedunder the appropriate conditions to allow the stem cells todifferentiate and populate the matrix. The tissues and organs obtainedby the methods of the invention may be used for a variety of purposes,including research and therapeutic purposes.

In accordance with the present invention, embryonic-like stem cells areobtained from a placenta which has been exsanguinated and perfused for aperiod of at least two to twenty four hours following expulsion from theuterus to remove all residual cells. The exsanguinated placenta is thencultured under the appropriate conditions to allow for the production ofendogenous stem cells originating from the placenta.

The methods of the present invention relate to the use of embryonic-likestem cells which have been originated from a placenta to seed a matrix.Once obtained from a cultured placenta, the embryonic-like stem cellsmay be characterized by a number of methods, including but not limitedto, immunochemistry, and the presence of particular cell surfacemarkers. Preferred stem cells to be used in accordance with the presentinvention may be identified by the presence of the following cellsurface markers: CD10+, CD29+, CD34−, CD44+, CD45−, CD54+, CD90+, SH2+,SH3+, SH4+, SSEA3−, SSEA4−, OCT−4+, and APC-p+.

In accordance with the methods of the present invention, the stem cellsmay be differentiated along specific cell lineages, includingadipogenic, chondrogenic, osteogenic, neurogenic and hepatogeniclineages. In another embodiment of the invention, it may be preferableto stimulate differentiation of embryonic-like stem cells to aparticular lineage prior to seeding the cells to a particular matrix. Inaccordance with this embodiment, cultured placentas may be stimulated toproduce cells of a particular lineage, by introducing into the placentaexogenous cells or tissues of the desired lineage. For example, prior toseeding embryonic-like stem cells on a matrix or decellularized organfor propagation and growth into liver tissue, the cultured placenta maybe stimulated to produce hepatogenic stem cells by introducing exogenoushepatogenic cells or tissue into the placenta.

The present invention also relates to the use of the cultured placentaas a bioreactor to stimulate the propagation of embryonic-like stemcells of a particular lineage. For example, the cultured placenta can bestimulated to produce embryonic-like stem cells which have becomecommitted to a particular lineage, including but not limited to,adipogenic, chondrogenic, osteogenic, neurogenic and hapatogic lineages.In accordance with this embodiment of the invention, the culturedplacenta may be stimulated to produce cells of a particular lineage, byexposing the cultured placenta to exogenous cells or tissues of thedesired lineage.

By way of example, and not by way of limitation, in order to generatecells of a hepatic lineage, the placenta may be exposed to liver cells.In accordance with this embodiment, liver cells are introduced into theplacenta by any number of methods, including injecting the liver cellsas a single cell suspension or as islands of cells into the vasculatureor directly into the placenta. Following introduction of the livercells, the placenta would be perfused in accordance with the methodsdescribed herein to allow for the recovery of hepatic stem cells fromthe placenta.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of the cannulation of the vein andartery of a placenta to perfuse the placenta and then collect theperfusate.

FIGS. 2 a-e are schematics showing the collection, clamping, perfusion,collection and storage of a drained and perfused placenta.

FIG. 3 is a cross-sectional schematic of a perfused placenta in a devicefor use as a bioreactor.

FIG. 4 is a selection scheme for sorting cells retrieved from a perfusedplacenta.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “bioreactor” refers to an ex vivo system forpropagating cells, producing or expressing biological materials andgrowing or culturing cells, tissues, organoids, viruses andmicroorganisms.

As used herein, the term “embryonic stem cell” refers to a cell that isderived from the inner cell mass of a blastocyst (e.g., a 4- to5-day-old human embryo) and that is pluripotent.

As used herein, the term “embryonic-like stem cell” refers to a cellthat is not derived from the inner cell mass of a blastocyst. As usedherein, an “embryonic-like stem cell” may also be referred to as a“placental stem cell.” An embryonic-like stem cell, however, may be apluripotent cell, a multipotent cell, or a committed progenitor cell.According to the methods of the invention, embryonic-like stem cellsderived from the placenta may be collected from the isolated placentaonce it has been exsanguinated and perfused for a period of timesufficient to remove residual cells.

As used herein, the term “exsanguinated” or “exsanguination,” when usedwith respect to the placenta, refers to the removal and/or draining ofsubstantially all cord blood from the placenta. In accordance with thepresent invention, exsanguination of the placenta can be achieved by,for example, but not by way of limitation, draining, gravity inducedefflux, massaging, squeezing, pumping, etc. Exsanguination of theplacenta may further be achieved by perfusing, rinsing or flushing theplacenta with a fluid that may or may not contain agents, such asanticoagulants, to aid in the exsanguination of the placenta.

As used herein, the term “perfuse” or “perfusion” refers to the act ofpouring or passaging a fluid over or through an organ or tissue,preferably the passage of fluid through an organ or tissue withsufficient force or pressure to remove any residual cells, e.g.,non-attached cells from the organ or tissue. As used herein, the term“perfusate” refers to the fluid collected following its passage throughan organ or tissue.

As used herein, the term “exogenous cell” refers to a “foreign” cell,i.e., a heterologous cell (i.e., a “non-self” cell derived from a sourceother than the placental donor) or autologous cell (i.e., a “self” cellderived from the placental donor) that is derived from an organ ortissue other than the placenta.

As used herein, the term “organoid” refers to an aggregation of one ormore cell types assembled in superficial appearance or in actualstructure as any organ or gland of a mammalian body, preferably thehuman body.

As used herein, the term “multipotent cell” refers to a cell that hasthe capacity to grow into any of subset of the mammalian body'sapproximately 260 cell types. Unlike a pluripotent cell, a multipotentcell does not have the capacity to form all of the cell types.

As used herein, the term “pluripotent cell” refers to a cell that hascomplete differentiation versatility, i.e., the capacity to grow intoany of the mammalian body's approximately 260 cell types. A pluripotentcell can be self-renewing, and can remain dormant or quiescent within atissue. Unlike a totipotent cell (e.g., a fertilized, diploid egg cell),an embryonic stem cell cannot usually form a new blastocyst.

As used herein, the term “progenitor cell” refers to a cell that iscommitted to differentiate into a specific type of cell or to form aspecific type of tissue.

As used herein, the term “stem cell” refers to a master cell that canreproduce indefinitely to form the specialized cells of tissues andorgans. A stem cell is a developmentally pluripotent or multipotentcell. A stem cell can divide to produce two daughter stem cells, or onedaughter stem cell and one progenitor (“transit”) cell, which thenproliferates into the tissue's mature, fully formed cells.

As used herein, the term “totipotent cell” refers to a cell that is ableto form a complete embryo (e.g., a blastocyst).

I. Tissue Matrices

Decellularized Tissue Matrices

A xenogeneic (or allogeneic) tissue matrix is processed to remove nativecells and other antigens and cellular debris from the decellularizedtissue matrix, and, optionally, treated to inhibit generation of newimmunological sites. Optionally, this tissue matrix can then be treatedwith the cellular adhesion factors described below to enhance attachmentof cells to the matrix during the process of repopulating the tissuematrix with such new cells. Different properties of the resulting matrixcan be obtained through the selection of cell types used to repopulatethe natural tissue matrices, such as the ability to synthesize proteinsotherwise atypical for the natural tissue at the site of implantation orunique to certain age groups. These hybrid grafts combine the structuraladvantages of bioprosthetic grafts with the functional and regenerativecapabilities of allografts as well as display attenuated or no immuneresponse, limited propensity to calcify, and little stimulation ofthromboembolism.

Depending on the type of transplant intended, if the recipient is human,the initial transplant tissue or organ may be of non-human origin. Thesetissues or organs may be obtained at approved slaughterhouses fromanimals fit for human consumption or from herds of domesticated animalsmaintained for the purpose of providing these tissues or organs. Thetissues or organs are handled in a sterile manner, and any furtherdissection of the tissue or organs is carried out under asepticconditions.

After collection and dissection, this tissue may be sterilized byincubating it in a sterile buffered nutrient solution containingantimicrobial agents, for example an antibacterial, an antifungal, or asterilant compatible with the transplant tissue. The sterilizedtransplant tissue may then be cryopreserved for further processing at alater time or may immediately be further processed according to the nextsteps of this process including a later cryopreservation of the tissuematrix or other tissue products of the process.

The tissue is first decellularized. Native viable cells as well as othercellular and acellular structures or components which may elicit anadverse immune response by the implant recipient are removed. Severalmeans of reducing the viability of native cells in tissues and organsare known, including physical, chemical, and biochemical methods. See,e.g. U.S. Pat. No. 5,192,312 (Orton) which is incorporated herein byreference. Such methods may be employed in accordance with the processdescribed herein. However, the decellularization technique employedshould not result in gross disruption of the anatomy of the transplanttissue or substantially alter the biomechanical properties of itsstructural elements. The treatment of the tissue to produce adecellularized tissue matrix should also not leave a cytotoxicenvironment that mitigates against subsequent repopulation of the matrixwith cells that are allogeneic or autologous to the recipient. Cells andtissues that are allogeneic to the recipient are those that originatewith or are derived from a donor of the same species as the recipient.Autologous cells or tissues are those that originate with or are derivedfrom the recipient.

Physical forces, for example the formation of intracellular ice, can beused to decellularize transplant tissues. For example, vapor phasefreezing (slow rate of temperature decline) of intact heart valves canreduce the cellularity of the heart valve leaflets as compared to liquidphase freezing (rapid). However, slow freezing processes, in the absenceof cryoprotectant, may result in tissue disruption such as the crackingof heart valve conduits. Colloid-forming materials may be added duringfreeze-thaw cycles to alter ice formation patterns in the tissue.Polyvinylpyrrolidone (10% w/v) and dialyzed hydroxyethyl starch (10%w/v) may be added to standard cryopreservation solutions (DMEM, 10%DMSO, 10% fetal bovine serum) to reduce extracellular ice formationwhile permitting formation of intracellular ice. This allows a measureof decellularization while providing the tissue matrix with someprotection from ice damage.

Alternatively, various enzymatic or other chemical treatments toeliminate viable native cells from implant tissues or organs may beused. For instance, extended exposure of cells to proteases such astrypsin result in cell death. However, because at least a portion of thetype I collagen molecule is sensitive to a variety of proteases,including trypsin, this may not be the approach of choice forcollagenous grafts intended for implant in high mechanical stresslocations.

Combinations of different classes of detergents, for example, a nonionicdetergent, Triton X-100, and an anionic detergent, sodium dodecylsulfate, may disrupt cell membranes and aid in the removal of cellulardebris from tissue. However, steps should be taken to eliminate anyresidual detergent levels in the tissue matrix, so as to avoidinterference with the later repopulating of the tissue matrix withviable cells.

The decellularization of the transplant tissue is preferablyaccomplished by the administration of a solution effective to lysenative cells in the transplant tissue. Preferably, the solution is anaqueous hypotonic or low ionic strength solution formulated toeffectively lyse the native tissue cells. Such an aqueous hypotonicsolution may be de-ionized water or an aqueous hypotonic buffer.Preferably the aqueous hypotonic buffer may contain additives thatprovide sub-optimal conditions for the activity of selected proteases,for example collagenase, which may be released as a result of cellularlysis. Additives such as metal ion chelators, for example1,10-phenanthroline and ethylenediaminetetraacetic acid (EDTA), createan environment unfavorable to many proteolytic enzymes. Providingsub-optimal conditions for proteases such as collagenase, may assist inprotecting the tissue matrix from degradation during the lysis step.Suboptimal conditions for proteases may be achieved by formulating thehypotonic lysis solution to eliminate or limit the amount of calcium andzinc ions available in solution. Many proteases are active in thepresence of calcium and zinc ions and lose much of their activity incalcium and zinc ion free environments. Preferably, the hypotonic lysissolution will be prepared selecting conditions of pH, reducedavailability of calcium and zinc ions, presence of metal ion chelatorsand the use of proteolytic inhibitors specific for collagenase such thatthe solution will optimally lyse the native cells while protecting theunderlying tissue matrix from adverse proteolytic degradation. Forexample a hypotonic lysis solution may include a buffered solution ofwater, pH 5.5 to 8, preferably pH 7 to 8, free from calcium and zincions and including a metal ion chelator such as EDTA. Additionally,control of the temperature and time parameters during the treatment ofthe tissue matrix with the hypotonic lysis solution, may also beemployed to limit the activity of proteases.

It is preferred that the decellularization treatment of the tissuematrix also limits the generation of new immunological sites. Whilecollagen is typically substantially non immunogenic, partial enzymaticdegradation of collagen may lead to heightened immunogenicity.Accordingly, a preferable step of this process includes treatment of thetissue with enzymes, such as nucleases, effective to inhibit cellularmetabolism, protein production and cell division without degrading theunderlying collagen matrix. Nucleases that can be used for digestion ofnative cell DNA and RNA include both exonucleases and endonucleases. Awide variety of which are suitable for use in this step of the processand are commercially available. For example, exonucleases thateffectively inhibit cellular activity include DNAase I (SIGMA ChemicalCompany, St. Louis, Mo.) and RNAase A (SIGMA Chemical Company, St.Louis, Mo.) and endonucleases that effectively inhibit cellular activityinclude EcoR I (SIGMA Chemical Company, St. Louis, Mo.) and Hind III(SIGMA Chemical Company, St. Louis, Mo.).

It is preferable that the selected nucleases are applied in aphysiological buffer solution which contains ions which are optimal forthe activity of the nuclease. Such ions include magnesium and calciumsalts. It is also preferred that the ionic concentration of the bufferedsolution, the treatment temperature and the length of treatment areselected to assure the desired level of effective nuclease activity. Thebuffer is preferably hypotonic to promote access of the nucleases totile cell interiors. For treatment of endogenous endothelial cells ofnon-human heart valve tissue, particularly valves of porcine or bovineorigin the tissue is preferably treated with a physiologically bufferedmedium comprised of nucleases DNAase I and RNAase A. Preferably, thenuclease degradation solution contains about 0.1 microgram/ml to 50microgram/ml, preferably 10 microgram/ml, of the nuclease DNAase I, and0.1 microgram/ml to 10 microgram/ml, preferably 1.0 microgram/ml, ofRNAase A. The tissue may be decellularized by application of theforegoing at a temperature of about 20 C to 38 C, preferably at about 37C (Centigrade), for about 30 minutes to 6 hours, while at the same timethe generation of new immunological sites as a result of collagendegradation is limited.

Other enzymatic digestions may be suitable for use herein, for example,enzymes that will disrupt the function of native cells in a transplanttissue may be used. For example, phospholipase, particularlyphospholipases A or C, in a buffered solution, may be used to inhibitcellular function by disrupting cellular membranes of endogenous cells.Preferably, the enzyme employed should not have a detrimental effect onthe tissue matrix protein. The enzymes suitable for use may also beselected with respect to inhibition of cellular integrity, and alsoinclude enzymes which may interfere with cellular protein production.The pH of the vehicle, as well as the composition of the vehicle, willalso be adjusted with respect to the pH activity profile of the enzymechosen for use. Moreover, the temperature applied during application ofthe enzyme to the tissue should be adjusted in order to optimizeenzymatic activity.

Following decellularization, the tissue matrix is washed to assureremoval of cell debris which may include cellular protein, cellularlipids, and cellular nucleic acid, as well as any extracellular debrissuch as extracellular soluble proteins, lipids and proteoglycans.Removal of this cellular and extracellular debris reduces the likelihoodof the transplant tissue matrix eliciting an adverse immune responsefrom the recipient upon implant. For example, the tissue may beincubated in a balanced salt solution such as Hanks' Balanced SaltSolution (HBSS). The composition of the balanced salt solution wash, andthe conditions under which it is applied to the transplant tissue matrixmay be selected to diminish or eliminate the activity of the nuclease orother enzyme utilized during the decellularization process. Such abalanced salt wash solution would preferably not contain magnesium orcalcium salts, and the washing process may include incubation at atemperature of between about 2 C and 42 C, with 4 C most preferable. Thetransplant tissue matrix may be incubated in the balanced salt washsolution for up to 10 to 12 days, with changes in wash solution everysecond or third day. Optionally, an antibacterial, an antifungal or asterilant or a combination thereof, may be included in the balanced saltwash solution to protect the transplant tissue matrix from contaminationwith environmental pathogens.

The tissue matrix can be preserved by cryopreservation. Techniques ofcryopreservation of tissue are well known in the art. Brockbank, K. G.M. Basic Principles of Viable Tissue Preservation. In: TransplantationTechniques and Use of Cryopreserved Allograft Cardiac Valves andVascular Tissue. D. R. Clarke (ed.), Adams Publishing Group, Ltd.,Boston. pp 9-23, discusses cryopreservation of tissues and organs and ishereby incorporated by reference.

The tissue matrix, whether or not having been cryopreserved, may be nexttreated to enhance the adhesion and inward migration of the allogeneicor autologous cells, in vitro, which will be used to repopulate thetransplant tissue.

The extent of attachment is increased by the addition of serum (human orfetal bovine, maximal binding with 1% serum) and by purified fibronectinto the culture medium. Each of the two homologous subunits offibronectin has two cell recognition regions, the most important ofwhich has the Arg-Gly-Asp (RGD) sequence. A second site, bindingglycosaminoglycans, acts synergistically and appears to stabilize thefibronectin-cell interactions mediated by the RGD sequence. Heparinsulfate along with chondroitin sulfate are the two glycosaminoglycansidentified on cell surfaces. Heparin sulfate is linked to core proteins(syndecan or hyaluronectin) which can either be integral or membranespanning. Cellular binding sites for extracellular matrix glycoproteinsare called integrins and these mediate tight binding of cells to theadhesion factors. Each adhesion factor appears to have a specializedintegrin although a single integrin may bind to several extracellularmatrix factors. Fibroblasts, when adherent to intact fibronectin (celland heparin-binding domains) display a contracted morphology with focaladhesions. Without the heparin binding domain, fibroblasts will spreadbut fail to develop focal adhesions.

Another method whereby cell attachment to the matrix is enhanced is byincubation of the decellularized tissue matrix in a nutrient solutioncontaining extracellular matrix protein such as fibronectin and aglycosaminoglycan for a period effective for binding of the fibronectinto surfaces of the transplant tissue matrix to be repopulated. Preferredbuffers for use with fibronectin/glycosaminoglycan include sodiumphosphate/glycerin/bovine serum albumin (Fetal Bovine Serum,BIO-WHITTAKER) and Dulbecco's Modified Eagle's Medium (DMEM), (GIBCO).These buffers typically are used to provide a physiological acceptablepH of about 7.0 to 7.6. The presence of the extracellular matrixproteins establish a surface on the tissue matrix to which the cellsthat have been chosen to repopulate the matrix attach. The stimulus ofthe extracellular matrix protein promotes cell repopulation in thegraft. A source of fibronectin is from human blood, processed to limitcontamination with virus. The preferred glycosaminoglycan is heparin.The concentration of glycoprotein used as the adhesion factor to treatthe tissue matrix may range from about 1 to about 100 microgram/ml, witha fibronectin concentration of 10 microgram/ml being preferred. Thepreferred weight ratio of fibronectin to heparin is about 10 partsfibronectin to about 1 part glycosaminoglycan, e.g. heparin. This isoptimal for repopulation of porcine heart valve leaflets, but may rangefrom about 0.1:1 to about 10:0.1 depending on the tissue used.

A variety of substances may be employed to enhance cell chemotaxis,increasing the rate of directional movement along a concentrationgradient of the substance in solution. With respect to fibroblast cells,fibroblast growth factor, platelet-derived growth factor, transforminggrowth factor-.beta., and the substrate-adhesion molecules, fibrillarcollagens, collagen fragments, and fibronectin are chemotactic forfibroblasts. In contrast to cell adhesion, fibroblast migration requiresde novo protein synthesis; protein synthesis in normal fibroblasticcells is stimulated by adhesion of cells to fibronectin, so theprocesses of cell adhesion and cell migration during repopulation arebelieved to be interrelated.

Synthetic Tissue Matrices

Tissue matrices can also be formed of synthetic or natural materials,such as collagen or polylactide-co-glycolide. A number of thesematerials are known. For example, a method for forming artificial skinby seeding a fibrous lattice with epidermal cells is described in U.S.Pat. No. 4,485,097 (Bell), which discloses a hydrated collagen latticethat, in combination with contractile agents such as platelets andfibroblasts and cells such as keratinocytes, is used to produce askin-like substance. U.S. Pat. No. 4,060,081 (Yannas et al.) discloses amultilayer membrane for use as a synthetic skin that is formed from aninsoluble modified collagen material that is very slowly degradable inthe presence of body fluids and enzymes. U.S. Pat. No. 4,458,678 (Yannaset al.) discloses a process for making a skin-like material wherein abiodegradable fibrous lattice formed from collagen cross-linked withglycosaminoglycan is seeded with epidermal cells.

U.S. Pat. No. 4,520,821 (Schmidt) describes a similar approach to makelinings to repair defects in the urinary tract. Epithelial cells areimplanted onto the surface of a liquid impermeable synthetic polymericmatrix where they form a monolayer lining on the matrix.

Vacanti, et al., “Selective cell transplantation using bioabsorbableartificial polymers as matrices” J. Pediat. Surg. 23, 3-9 (1988) andVacanti, “Beyond Transplantation” Arch. Surg. 123, 545-549 (1988),describe an approach for making new organs for transplantation. Vacanti,et al., recognized that cells require a matrix for attachment andsupport if they are to survive following implantation, that a minimumnumber of cells was essential for function in vivo, and that the matrixmust be porous enough to allow nutrients and gases to reach all of thecells on and within the matrix by diffusion, until the matrix-cellstructure was vascularized. They report that there are advantages tousing synthetic biodegradable polymer substrates to form a scaffold thatmimics its natural counterparts, the extracellular matrices (ECM) of thebody, serving as both a physical support and an adhesive substrate forisolated parenchymal cells during in vitro culture, and subsequentimplantation, degrading as the cells begin to secrete they own ECMsupport. These matrices have also been implanted and seeded directly, toform new tissues.

II. Cells to be Seeded onto/into Decellularized Tissues

Human stem cells are totipotential or pluripotential precursor cellscapable of generating a variety of mature human cell lineages. Thisability serves as the basis for the cellular differentiation andspecialization necessary for organ and tissue development. Recentsuccess at transplanting such stem cells have provided new clinicaltools to reconstitute and/or supplement the bone marrow aftermyeloablation due to disease, exposure to toxic chemical or radiation.Further evidence exists which demonstrates that stem cells can beemployed to repopulate many, if not all, tissues and restore physiologicand anatomic functionality. The application of stem cells in tissueengineering, gene therapy delivery and cell therapeutics is alsoadvancing rapidly.

Obtaining sufficient human stem cells has been problematic for severalreasons. First, isolation of normally occurring populations of stemcells in adult tissues has been technically difficult, costly and verylimited in quantity. Secondly, procurement of these cells from embryosor fetal tissue including abortuses has raised many ethical and moralconcerns. The widely held belief that the human embryo and fetusconstitute independent life has justified a moratorium on the use ofsuch sources for any purpose. Alternative sources which do not violatethe sanctity of independent life are essential for further progress inthe use of stem cells clinically.

Umbilical cord blood (cord blood) is a known source of hemopoieticpluripotent, progenitor stem cells that are cryopreserved for use inhemopoietic reconstitution. The use of cord blood for this purpose iswell known and is becoming a widely used therapeutic procedure. Theconventional technique for the collection of cord blood is based on theuse of a needle or cannula which is used with the aid of gravity todrain the cord blood from the placenta. Usually the needle or cannula isplaced in the umbilical vein and the placenta is gently massaged to aidin draining the cord blood from the placenta.

The Applicant has unexpectedly discovered that the placenta after birthcontains quiescent cells which can be activated if the placenta isproperly processed after birth. For example, after expulsion from thewomb, the placenta is exsanguinated as quickly as possible to prevent orminimize apoptosis. Subsequently, as soon as possible afterexsanguination the placenta is perfused to remove blood, residual cells,proteins, factors and any other materials present in the organ.Perfusion is normally continued with an appropriate perfusate for atleast two to more than twenty-four hours. In several additionalembodiments the placenta is perfused for at least 4, 6, 8, 10, 12, 14,16, 18, 20, and 22 hours. In other words, this invention is based atleast in part on the discovery that the cells of a post-partum placentacan be activated by exsanguination and perfusion for a sufficient amountof time. Therefore, the placenta can readily be used as a rich andabundant source of human placental stem cells, which cells can be usedfor research, including drug discovery, treatment and prevention ofdiseases, in particular transplantation surgeries or therapies, and thegeneration of committed cells, tissues and organoids.

Further, surprisingly and unexpectedly the human placental stem cellsproduced by the exsanguinated, perfused and/or cultured placenta arepluripotent stem cells that can readily be differentiated into anydesired cell type.

The present invention relates to methods of treating and culturing anisolated placenta for use as a bioreactor for the production andpropagation of embryonic-like stem cells originating from the placentaor from exogenous sources. The present invention also relates to the useof a cultured placenta as a bioreactor to produce biological materials,including, but not limited to, antibodies, hormones, cytokines, andgrowth factors. The present invention also relates to methods ofcollecting and isolating the stem cells and biological materials fromthe cultured placenta.

The present invention relates to methods of perfusing and exsanguinatingan isolated placenta once it has been expunged from a uterus, to removeall residual cells. The invention further relates to culturing theisolated and exsanguinated placenta under the appropriate conditions toallow for the production and propagation of embryonic-like stem cells.

The present invention provides a method of extracting and recoveringembryonic-like stem cells, including, but not limited to pluripotent ormultipotent stem cells, froth an exsanguinated human placenta.Embryonic-like stem cells have characteristics of embryonic stem cellsbut are not derived from the embryo. Such cells are as versatile (e.g.,pluripotent) as human embryonic stem cells. According to the methods ofthe invention, human placenta is used post-birth as the source ofembryonic-like stem cells.

According to the methods of the invention embryonic-like stem cells areextracted from a drained placenta by means of a perfusion technique thatutilizes either or both of the umbilical artery and umbilical vein. Theplacenta is preferably drained by exsanguination and collection ofresidual blood (e.g., residual umbilical cord blood). The drainedplacenta is then processed in such a manner as to establish an ex vivo,natural bioreactor environment in which resident embryonic-like stemcells within the parenchyma and extravascular space are recruited. Theembryonic-like stem cells migrate into the drained, emptymicrocirculation where, according to the methods of the invention, theyare collected, preferably by washing into a collecting vessel byperfusion.

Methods of Isolating and Culturing Placenta

The following discloses, among other things, the method of collectingplacental stem cells and other multipotent stem cells from a placenta.The present applicant describes this method in detail in applicant'spending U.S. patent application Ser. No. 10/004,942, filed Dec. 5, 2001,claiming priority to U.S. Provisional Patent Application No. 60/251,900,filed Dec. 6, 2000, and wherein pending U.S. patent application Ser. No.10/004,942 is incorporated herein by reference in this application.

Pretreatment of Placenta

According to the methods of the invention, a placenta (for example, ahuman placenta) is recovered shortly after its expulsion after birthand, in certain embodiments, the cord blood in the placenta isrecovered. In certain embodiments, the placenta is subjected to aconventional cord blood recovery process. Such cord blood recovery maybe obtained commercially, such as for example Lifebank Services,Bethesda, Md. The cord blood can be drained shortly after expulsion ofthe placenta. Alternatively, the placenta can be stored, preferably forno longer than 48 hours, prior to the collection of cord blood.

The placenta is preferably recovered after expulsion under asepticconditions, and stored in an anticoagulant solution at a temperature of5 to 25 degrees C. (centigrade). Suitable anticoagulant solutions arewell known in the art. For example, a solution of heparin or warfarinsodium can be used. In a preferred embodiment, the anticoagulantsolution comprises a solution of heparin (1% w/w in 1:1000 solution).The drained placenta is preferably stored for no more than 36 hoursbefore the embryonic-like stem cells are collected. The solution whichis used to perfuse the placenta to remove residual cells can be the samesolution used to perfuse and culture the placenta for the recovery ofstem cells. Any of these perfusates may be collected and used as asource of embryonic-like stem cells.

In certain embodiments, the proximal umbilical cord is clamped,preferably within 4-5 cm (centimeter) of the insertion into theplacental disc prior to cord blood recovery. In other embodiments, theproximal umbilical cord is clamped after cord blood recovery but priorto further processing of the placenta.

Conventional techniques for the collection of cord blood may be used.Typically a needle or cannula is used, with the aid of gravity, to draincord blood from (i.e., exsanguinate) the placenta (Boyse et al., U.S.Pat. No. 5,192,553, issued Mar. 9, 1993; Anderson, U.S. Pat. No.5,372,581, entitled Method and apparatus for placental blood collection,issued Dec. 13, 1994; Hessel et al., U.S. Pat. No. 5,415,665, entitledUmbilical cord clamping, cutting, and blood collecting device andmethod, issued May 16, 1995). The needle or cannula is usually placed inthe umbilical vein and the placenta is gently massaged to aid indraining cord blood from the placenta.

Typically, a placenta is transported from the delivery or birthing roomto another location, e.g., a laboratory, for recovery of the cord bloodand/or drainage and perfusion. The placenta is preferably transported ina sterile, thermally insulated transport device (maintaining thetemperature of the placenta between 20-28EC), for example, by placingthe placenta, with clamped proximal umbilical cord, in a sterilezip-lock plastic bag, which is then placed in an insulated container, asshown in FIGS. 2 a-e.

In a preferred embodiment, the placenta is recovered from a patient byinformed consent and a complete medical history of the patient prior to,during and after pregnancy is also taken and is associated with theplacenta. These medical records can be used to coordinate subsequent useof the placenta or the stem cells harvested therefrom. For example, thehuman placental stem cells can then easily be used for personalizedmedicine for the infant in question, the parents, siblings or otherrelatives. Indeed, the human placental stem cells are more versatilethan cord blood. However, it should be noted that the invention includesthe addition of human placental stem cells produced by theexsanguinated, perfused and/or cultured placenta to cord blood from thesame or different placenta and umbilical cord. The resulting cord bloodwill have an increased concentration/population of human stem cells andthereby is more useful for transplantation e.g. for bone marrowtransplantations.

Exsanguination of Placenta and Removal of Residual Cells

The invention provides a method for recovery of stem or progenitorcells, including, but not limited to embryonic-like stem cells, from aplacenta that is exsanguinated, i.e., completely drained of the cordblood remaining after birth and/or a conventional cord blood recoveryprocedure. According to the methods of the invention, the placenta isexsanguinated and perfused with a suitable aqueous perfusion fluid, suchas an aqueous isotonic fluid in which an anticoagulant (e.g., heparin,warfarin sodium) is dissolved. Such aqueous isotonic fluids forperfusion are well known in the art, and include, e.g., a 0.9 N sodiumchloride solution. The perfusion fluid preferably comprises theanticoagulant, such as heparin or warfarin sodium, at a concentrationthat is sufficient to prevent the formation of clots of any residualcord blood. In a specific embodiment, a concentration of from 100 to1000 units of heparin is employed. In one embodiment, apoptosisinhibitors can be used during and immediately after exsanguination andthen these agents can be washed from the placenta.

According to the methods of the invention, the placenta is exsanguinatedby passage of the perfusion fluid through either or both of theumbilical artery and umbilical vein, using a gravity flow into theplacenta. The placenta is preferably oriented (e.g., suspended) in sucha manner that the umbilical artery and umbilical vein are located at thehighest point of the placenta. In a preferred embodiment, the umbilicalartery and the umbilical vein are connected simultaneously, as shown inFIG. 1, to a pipette that is connected via a flexible connector to areservoir of the perfusion fluid. The perfusion fluid is passed into theumbilical vein and artery and collected in a suitable open vessel fromthe surface of the placenta that was attached to the uterus of themother during gestation.

In a preferred embodiment, the proximal umbilical cord is clamped duringperfusion, and more preferably, is clamped within 4-5 cm (centimeter) ofthe cord's insertion into the placental disc.

In one embodiment, a sufficient amount of perfusion fluid is used thatwill result in removal of all residual cord blood and subsequentcollection or recovery of placental cells, including but not limited toembryonic-like stem cells and progenitor cells, that remain in theplacenta after removal of the cord blood.

It has been observed that when perfusion fluid is first collected from aplacenta during the exsanguination process, the fluid is colored withresidual red blood cells of the cord blood. The perfusion fluid tends tobecome clearer as perfusion proceeds and the residual cord blood cellsare washed out of the placenta. Generally from 30 to 100 ml (milliliter)of perfusion fluid is adequate to exsanguinate the placenta and torecover an initial population of embryonic-like cells from the placenta,but more or less perfusion fluid may be used depending on the observedresults.

Culturing Placenta

After exsanguination and perfusion of the placenta, the embryonic-likestem cells are observed to migrate into the exsanguinated and perfusedmicrocirculation of the placenta where, according to the methods of theinvention, they are collected, preferably by washing into a collectingvessel by perfusion. Perfusing the isolated placenta not only serves toremove residual cord blood but also provide the placenta with theappropriate nutrients, including oxygen.

In certain embodiments of the invention, the drained, exsanguinatedplacenta is cultured as a bioreactor, i.e., an ex vivo system forpropagating cells or producing biological materials. The number ofpropagated cells or level of biological material produced in theplacental bioreactor is maintained in a continuous state of balancedgrowth by periodically or continuously removing a portion of a culturemedium or perfusion fluid that is introduced into the placentalbioreactor, and from which the propagated cells or the producedbiological materials may be recovered. Fresh medium or perfusion fluidis introduced at the same rate or in the same amount.

The number and type of cells propagated may easily be monitored bymeasuring changes in morphology and cell surface markers using standardcell detection techniques such as flow cytometry, cell sorting,immunocytochemistry (e.g., staining with tissue specific or cell-markerspecific antibodies), fluorescence activated cell sorting (FACS),magnetic activated cell sorting (MACS), by examination of the morphologyof cells using light or confocal microscopy, or by measuring changes ingene expression using techniques well known in the art, such as PCR andgene expression profiling.

In one embodiment, the cells may be sorted using a fluorescenceactivated cell sorter (FACS). Fluorescence activated cell sorting (FACS)is a well-known method for separating particles, including cells, basedon the fluorescent properties of the particles (Kamarch, 1987, MethodsEnzymol, 151:150-165). Laser excitation of fluorescent moieties in theindividual particles results in a small electrical charge allowingelectromagnetic separation of positive and negative particles from amixture. In one embodiment, cell surface marker-specific antibodies orligands are labeled with distinct fluorescent labels. Cells areprocessed through the cell sorter, allowing separation of cells based ontheir ability to bind to the antibodies used. FACS sorted particles maybe directly deposited into individual wells of 96-well or 384-wellplates to facilitate separation and cloning.

In another embodiment, magnetic beads can be used to separate cells. Thecells may be sorted using a magnetic activated cell sorting (MACS)technique, a method for separating particles based on their ability tobind magnetic beads (0.5-100 μm diameter). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of antibody which specifically recognizes a cell-solidphase surface molecule or hapten. A magnetic field is then applied, tophysically manipulate the selected beads. The beads are then mixed withthe cells to allow binding. Cells are then passed through a magneticfield to separate out cells having cell surface markers. These cells canthen isolated and re-mixed with magnetic beads coupled to an antibodyagainst additional cell surface markers. The cells are again passedthrough a magnetic field, isolating cells that bound both theantibodies. Such cells can then be diluted into separate dishes, such asmicrotiter dishes for clonal isolation.

In preferred embodiments, the placenta to be used as a bioreactor isexsanguinated and washed under sterile conditions so that any adherentcoagulated and non-adherent cellular contaminants are removed. Theplacenta is then cultured or cultivated under aseptic conditions in acontainer or other suitable vessel, and perfused with perfusate solution(e.g., a normal saline solution such as phosphate buffered saline (PBS))with or without an anticoagulant (e.g., such as for example, heparin orwarfarin sodium), and/or with or without an antimicrobial agent (e.g.,such as antibiotics).

The effluent perfusate comprises both circulated perfusate, which hasflowed through the placental circulation, and extravasated perfusate,which exudes from or passes through the walls of the blood vessels intothe surrounding tissues of the placenta. The effluent perfusate iscollected, and preferably, both the circulated and extravasatedperfusates are collected, preferably in a sterile receptacle.Alterations in the conditions in which the placenta is maintained andthe nature of the perfusate can be made to modulate the volume andcomposition of the effluent perfusate.

Cell types are then isolated from the collected perfusate by employingtechniques known by those skilled in the art, such as for example, butnot limited to density gradient centrifugation, magnet cell separation,flow cytometry, affinity cell separation or differential adhesiontechniques.

In one embodiment, a placenta is placed in a sterile basin and washedwith 500 ml of phosphate-buffered normal saline. The wash fluid is thendiscarded. The umbilical vein is then cannulated with a cannula, e.g., aTEFLON7 or plastic cannula, that is connected to a sterile connectionapparatus, such as sterile tubing. The sterile connection apparatus isconnected to a perfusion manifold, as shown in FIG. 3. The containercontaining the placenta is then covered and the placenta is maintainedat room temperature (20-25 degrees C.) for a desired period of time,preferably from 2 to 24 hours, and preferably, no longer than 48 hours.The placenta may be perfused continually, with equal volumes ofperfusate introduced and effluent perfusate removed or collected.Alternatively, the placenta may be perfused periodically, e.g., forexample, at every 2 hours or at 4, 8, 12, and 24 hours, with a volume ofperfusate, e.g., preferably, 100 ml of perfusate (sterile normal salinesupplemented with or without 1000 u/l heparin and/or EDTA and/or CPDA(creatine phosphate dextrose)). In the case of periodic perfusion,preferably equal volumes of perfusate are introduced and removed fromthe culture environment of the placenta, so that a stable volume ofperfusate bathes the placenta at all times.

The effluent perfusate that escapes the placenta, e.g., at the oppositesurface of the placenta, is collected and processed to isolateembryonic-like stem cells, progenitor cells or other cells of interest.

Various media may be used as perfusion fluid for culturing orcultivating the placenta, such as DMEM, F-12, M199, RPMI, Fisher's,Iscore's, McCoy's and combinations thereof, supplemented with fetalbovine serum (FBS), whole human serum (WHS), or human umbilical cordserum collected at the time of delivery of the placenta.

In certain embodiments, the embryonic-like stem cells are induced topropagate in the placenta bioreactor by introduction of nutrients,hormones, vitamins, growth factors, or any combination thereof, into theperfusion solution. Serum and other growth factors may be added to thepropagation perfusion solution or medium. Growth factors are usuallyproteins and include for example, but are not limited to: cytokines,lymphokines, interferons, colony stimulating factors (CSF's),interferons, chemokines, and interleukins. Other growth factors that maybe used include recombinant human hematopoietic growth factorsincluding, for example, ligands, stem cell factors, thrombopoeitin(Tpo), interleukins, and granulocyte colony-stimulating factor (G-CSF).

The growth factors introduced into the perfusion solution can stimulatethe propagation of undifferentiated embryonic-like stem cells, committedprogenitor cells, or differentiated cells (e.g., differentiatedhematopoietic cells). The growth factors can stimulate the production ofbiological materials and bioactive molecules including for example, butnot limited to, immunoglobulins, hormones, enzymes or growth factors aspreviously described.

In one embodiment of the invention, the placenta is used as a bioreactorfor propagating endogenous cells (i.e., cells that originate from theplacenta), including but not limited to, various kinds of pluripotentand/or totipotent embryonic-like stem cells and lymphocytes. To use theplacenta as a bioreactor, it may be cultured for varying periods of timeunder sterile conditions by perfusion with perfusate solution asdisclosed herein. In specific embodiments, the placenta is cultured forat least 12, 24, 36, or 48 hours, or for 3-5 days, 6-10 days, or for oneto two weeks. In a preferred embodiment, the placenta is cultured for 48hours. The cultured placenta should be “fed” periodically to remove thespent media, depopulate released cells, and add fresh media. Thecultured placenta should be stored under sterile conditions to reducethe possibility of contamination, and maintained under intermittent andperiodic pressurization to create conditions that maintain an adequatesupply of nutrients to the cells of the placenta. It should berecognized that the perfusing and culturing of the placenta can be bothautomated and computerized for efficiency and increased capacity.

In another embodiment, the placenta is processed to remove allendogenous proliferating cells, such as embryonic-like stem cells, andto allow foreign (i.e., exogenous) cells to be introduced and propagatedin the environment of the perfused placenta. The invention contemplatesa large variety of stem or progenitor cells that can be cultured in theplacental bioreactor, including for example, but not limited to,embryonic-like stem cells, mesenchymal stem cells, stromal cells,endothelial cells, hepatocytes, keratinocytes, and stem or progenitorcells for a particular cell type, tissue or organ, including forexample, but not limited to neurons, myelin, muscle, blood, bone marrow,skin, heart, connective tissue, lung, kidney, liver, and pancreas (e.g.,pancreatic islet cells).

Sources of mesenchymal stem cells include bone marrow, embryonic yolksac, placenta, umbilical cord, fetal and adolescent skin, and blood.Bone marrow cells may be obtained from iliac crest, femora, tibiae,spine, rib or other medullary spaces.

Methods for the selective destruction, ablation or removal ofproliferating or rapidly dividing cells from a tissue or organ arewell-known in the art, e.g., methods of cancer or tumor treatment. Forexample, the perfused placenta may be irradiated with electromagnetic,UV, X-ray, gamma- or beta-radiation to eradicate all remaining viable,endogenous cells. The foreign cells to be propagated are introduced intothe irradiated placental bioreactor, for example, by perfusion.

Collection of Cells from the Placenta

As disclosed above, after exsanguination and perfusion of the placenta,embryonic-like stem cells migrate into the drained, emptymicrocirculation where, according to the methods of the invention, theyare collected, preferably by collecting the effluent perfusate in acollecting vessel.

In preferred embodiments, cells cultured in the placenta are isolatedfrom the effluent perfusate using techniques known by those skilled inthe art, such as, for example, density gradient centrifugation, magnetcell separation, flow cytometry, or other cell separation or sortingmethods well known in the art, and sorted, for example, according to thescheme shown in FIG. 4.

In a specific embodiment, cells collected from the placenta arerecovered from the effluent perfusate by centrifugation at X 00×g for 15minutes at room temperature, which separates cells from contaminatingdebris and platelets. The cell pellets are resuspended in IMDMserum-free medium containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL,NY). The total mononuclear cell fraction was isolated using Lymphoprep(Nycomed Pharma, Oslo, Norway) according to the manufacturer'srecommended procedure and the mononuclear cell fraction was resuspended.Cells were counted using a hemocytometer. Viability was evaluated bytrypan blue exclusion. Isolation of cells is achieved by “differentialtrypsinization”, using a solution of 0.05% trypsin with 0.2% EDTA(Sigma, St. Louis Mo.). Differential trypsinization was possible becausefibroblastoid cells detached from plastic surfaces within about fiveminutes whereas the other adherent populations required more than 20-30minutes incubation. The detached fibroblastoid cells were harvestedfollowing trypsinization and trypsin neutralization, using TrypsinNeutralizing Solution (TNS, BioWhittaker). The cells were washed inH.DMEM and resuspended in MSCGM.

In another embodiment, the isolated placenta is perfused for a period oftime without collecting the perfusate, such that the placenta may beperfused for 2, 4, 6, 8, 10, 12, 20 and 24 hours or even days before theperfusate is collected.

In another embodiment, cells cultured in the placenta bioreactor areisolated from the placenta by physically dissecting the cells away fromthe placenta.

In another embodiment, cells cultured in the placenta bioreactor areisolated from the placenta by dissociating the tissues of the placentaor a portion thereof, and recovering the cultured cells by art-knowncell separation or sorting methods such as density gradientcentrifugation, magnet cell separation, flow cytometry, etc.

In a preferred embodiment, perfusion of the placenta and collection ofeffluent perfusate is repeated once or twice during the culturing of theplacenta, until the number of recovered nucleated cells falls below 100cells/ml. The perfusates are pooled and subjected to lightcentrifugation to remove platelets, debris and de-nucleated cellmembranes. The nucleated cells are then isolated by Ficoll-Hypaquedensity gradient centrifugation and after washing, resuspended inH.DMEM. For isolation of the adherent cells, aliquots of 5-10×10⁶ cellsare placed in each of several T-75 flasks and cultured with commerciallyavailable Mesenchymal Stem Cell Growth Medium (MSCGM) obtained fromBioWhittaker, and placed in a tissue culture incubator (37EC, 5% CO₂).After 10 to 15 days, non-adherent cells are removed from the flasks bywashing with PBS. The PBS is then replaced by MSCGM. Flasks arepreferably examined daily for the presence of various adherent celltypes and in particular, for identification and expansion of clusters offibroblastoid cells.

In other embodiments, the cells collected from the placenta arecryopreserved for use at a later time. Methods for cryopreservation ofcells, such as stem cells, are well known in the art, for example,cryopreservation using the methods of Boyse et al. (U.S. Pat. No.5,192,553, issued Mar. 9, 1993) or Hu et al. (WO 00/73421, publishedDec. 7, 2000).

Cell Populations Obtained from or Cultured in Placenta

Embryonic-like stem cells obtained in accordance with the methods of theinvention may include pluripotent cells, i.e., cells that have completedifferentiation versatility, that are self-renewing, and can remaindormant or quiescent within tissue. The embryonic-like stem cells canalso include multipotent cells, committed progenitor cells, orfibroblastoid cells.

In a preferred embodiment, embryonic-like stem cells obtained by themethods of the invention are viable, quiescent, pluripotent stem cellsthat exist within a full-term human placenta and that can be recoveredfollowing successful birth and placental expulsion, resulting in therecovery of as many as one billion nucleated cells, which yield 50-100million multipotent and pluripotent stem tells.

The human placental stem cells provided by the placenta are surprisinglyembryonic-like, for example, the presence of the following cell surfacemarkers have been identified for these cells: SSEA3−, SSEA4−, OCT-4⁺ andABC-p⁺. Thus, the invention encompasses stem cells which have not beenisolated or otherwise obtained from an embryonic source but which can beidentified by the following markers: SSAE3−, SSAE4−, OCT-4+ and ABC-p+.In one embodiment, the human placental stem cells do not express MHCClass 2 antigens.

The stem cells isolated from the placenta are homogenous, and sterile.Further, the stem cells are readily obtained in a form suitable foradministration to humans, i.e., they are of pharmaceutical grade.

Preferred embryonic-like stem cells obtained by the methods of theinvention may be identified by the presence of the following cellsurface markers: CD10+, CD29+, CD34−, CD44+, CD45−, CD54+, CD90+, SH2+,SH3+, SH4+, SSEA3−, SSEA4−, OCT-4+, and ABC-p+. Such cell surfacemarkers are routinely determined according to methods well known in theart, e.g. by flow cytometry, followed by washing and staining with ananti-cell surface marker antibody. For example, to determine thepresence of CD-34 or CD-38, cells may be washed in PBS and thendouble-stained with anti-CD34 phycoerythrin and anti-CD38 fluoresceinisothiocyanate (Becton Dickinson, Mountain View, Calif.).

The embryonic-like stem cells obtained by the methods of the inventionmay be induced to differentiate along specific cell lineages, includingadipogenic, chondrogenic, osteogenic, hematopoietic, myogenic,vasogenic, neurogenic, and hepatogenic. In certain embodiments,embryonic-like stem cells obtained according to the methods of theinvention are induced to differentiate for use in transplantation and exvivo treatment protocols

In certain embodiments, embryonic-like stem cells obtained by themethods of the invention are induced to differentiate into a particularcell type and genetically engineered to provide a therapeutic geneproduct. In a specific embodiment, embryonic-like stem cells obtained bythe methods of the invention are incubated with a compound in vitro thatinduces it to differentiate, followed by direct transplantation of thedifferentiated cells to a subject. Thus, the invention encompassesmethods of differentiating the human placental stem cells using standardculturing media. Further, the invention encompasses hematopoietic cells,neuron cells, fibroblast cells, strand cells, mesenchymal cells andhepatic cells.

Embryonic-like stem cells may also be further cultured after collectionfrom the placenta using methods well known in the art, for example, byculturing on feeder cells, such as irradiated fibroblasts, obtained fromthe same placenta as the embryonic-like stem cells or from other humanor nonhuman sources, or in conditioned media obtained from cultures ofsuch feeder cells, in order to obtain continued long-term cultures ofembryonic-like stem cells. The embryonic-like stem cells may also beexpanded, either within the placenta before collection from theplacental bioreactor or in vitro after recovery from the placenta. Incertain embodiments, the embryonic-like stem cells to be expanded areexposed to, or cultured in the presence of, an agent that suppressescellular differentiation. Such agents are well-known in the art andinclude, but are not limited to, human Delta-1 and human Serrate-1polypeptides (see, Sakano et al., U.S. Pat. No. 6,337,387 entitled“Differentiation-suppressive polypeptide”, issued Jan. 8, 2002),leukemia inhibitory factor (LIF) and stem cell factor. Methods for theexpansion of cell populations are also known in the art (see e.g.,Emerson et al., U.S. Pat. No. 6,326,198 entitled “Methods andcompositions for the ex vivo replication of stem cells, for theoptimization of hematopoietic progenitor cell cultures, and forincreasing the metabolism, GM-CSF secretion and/or IL-6 secretion ofhuman stromal cells”, issued Dec. 4, 2001; Kraus et al., U.S. Pat. No.6,338,942, entitled “Selective expansion of target cell populations”,issued Jan. 15, 2002).

The embryonic-like stem cells may be assessed for viability,proliferation potential, and longevity using standard techniques knownin the art, such as trypan blue exclusion assay, fluorescein diacetateuptake assay, propidium iodide uptake assay (to assess viability); andthymidine uptake assay, MTT cell proliferation assay (to assessproliferation). Longevity may be determined by methods well known in theart, such as by determining the maximum number of population doubling inan extended culture.

In certain embodiments, the differentiation of stem cells or progenitorcells that are cultivated in the exsanguinated, perfused and/or culturedplacenta is modulated using an agent or pharmaceutical compositionscomprising a dose and/or doses effective upon single or multipleadministration, to exert an effect sufficient to inhibit, modulateand/or regulate the differentiation of a cell collected from theplacenta.

Agents that can induce stem or progenitor cell differentiation are wellknown in the art and include, but are not limited to, Ca²⁺, EGF, aFGF,bFGF, PDGF, keratinocyte growth factor (KGF), TGF-β, cytokines (e.g.,IL-1α, IL-1β, IFN-γ, TFN), retinoic acid, transferrin, hormones (e.g.,androgen, estrogen, insulin, prolactin, triiodothyronine,hydrocortisone, dexamethasone), sodium butyrate, TPA, DMSO, NMF, DMF,matrix elements (e.g., collagen, laminin, heparan sulfate, MatrigelJ),or combinations thereof. In addition, compounds may be used to modulatedifferentiation of cells collected from the placenta.

Agents that suppress cellular differentiation are also well-known in theart and include, but are not limited to, human Delta-1 and humanSerrate-1 polypeptides (see, Sakano et al., U.S. Pat. No. 6,337,387entitled “Differentiation-suppressive polypeptide”, issued Jan. 8,2002), leukemia inhibitory factor (LIF), and stem cell factor.

The agent used to modulate differentiation can be introduced into theplacental bioreactor to induce differentiation of the cells beingcultured in the placenta. Alternatively, the agent can be used tomodulate differentiation in vitro after the cells have been collected orremoved from the placenta.

Determination that a stem cell has differentiated into a particular celltype may be accomplished by methods well-known in the art, e.g.,measuring changes in morphology and cell surface markers usingtechniques such as flow cytometry or immunocytochemistry (e.g., stainingcells with tissue-specific or cell-marker specific antibodies), byexamination of the morphology of cells using light or confocalmicroscopy, or by measuring changes in gene expression using techniqueswell known in the art, such as PCR and gene-expression profiling.

In another embodiment, the cells cultured in the placenta are stimulatedto produce bioactive molecules, such as immunoglobulins, hormones,enzymes.

In another embodiment, the cells cultured in the placenta are stimulatedto proliferate, for example, by administration of erythropoietin,cytokines, lymphokines, interferons, colony stimulating factors (CSF's),interferons, chemokines, interleukins, recombinant human hematopoieticgrowth factors including ligands, stem cell factors, thrombopoeitin(Tpo), interleukins, and granulocyte colony-stimulating factor (G-CSF)or other growth factors.

In another embodiment, cells cultured in the placenta are geneticallyengineered either prior to, or after collection from, the placenta,using, for example, a viral vector such as an adenoviral or retroviralvector, or by using mechanical means such as liposomal or chemicalmediated uptake of the DNA.

A vector containing a transgene can be introduced into a cell ofinterest by methods well known in the art, e.g., transfection,transformation, transduction, electroporation, infection,microinjection, cell fusion, DEAE dextran, calcium phosphateprecipitation, liposomes, LIPOFECTIN, lysosome fusion, syntheticcationic lipids, use of a gene gun or a DNA vector transporter, suchthat the transgene is transmitted to daughter cells, e.g., the daughterembryonic-like stem cells or progenitor cells produced by the divisionof an embryonic-like stem cell. For various techniques fortransformation or transfection of mammalian cells, see Keown et al.,1990, Methods Enzymol. 185: 527-37; Sambrook et al., 2001, MolecularCloning, A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press, N.Y.

Preferably, the transgene is introduced using any technique, so long asit is not destructive to the cell's nuclear membrane or other existingcellular or genetic structures. In certain embodiments, the transgene isinserted into the nucleic genetic material by microinjection.Microinjection of cells and cellular structures is commonly known andpracticed in the art.

For stable transfection of cultured mammalian cells, such as cellsculture in a placenta, only a small fraction of cells may integrate theforeign DNA into their genome. The efficiency of integration dependsupon the vector and transfection technique used. In order to identifyand select integrants, a gene that encodes a selectable marker (e.g.,for resistance to antibiotics) is generally introduced into the hostembryonic-like stem cell along with the gene sequence of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die). Such methods are particularlyuseful in methods involving homologous recombination in mammalian cells(e.g., in embryonic-like stem cells) prior to introduction ortransplantation of the recombinant cells into a subject or patient.

A number of selection systems may be used to select transformed hostembryonic-like cells. In particular, the vector may contain certaindetectable or selectable markers. Other methods of selection include butare not limited to selecting for another marker such as: the herpessimplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223),hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski,1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78: 2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147).

The transgene may integrate into the genome of the cell of interest,preferably by random integration. In other embodiments the transgene mayintegrate by a directed method, e.g., by directed homologousrecombination (“knock-in”), Chappel, U.S. Pat. No. 5,272,071; and PCTpublication No. WO 91/06667, published May 16, 1991; U.S. Pat. No.5,464,764; Capecchi et al., issued Nov. 7, 1995; U.S. Pat. No.5,627,059, Capecchi et al. issued, May 6, 1997; U.S. Pat. No. 5,487,992,Capecchi et al., issued Jan. 30, 1996).

In a specific embodiment, the methods of Bonadio et al. (U.S. Pat. No.5,942,496, entitled Methods and compositions for multiple gene transferinto bone cells, issued Aug. 24, 1999 and PCT WO95/22611, entitled“Methods and compositions for stimulating bone cells”, published Aug.24, 1995) are used to introduce nucleic acids into a cell of interest,such as a stem cell or progenitor cell cultured in the placenta, e.g.,bone progenitor cells.

Uses of Cultured Placenta as a Bioreactor

Exsanguinated and/or cultured placental cells can be used as abioreactor for the cultivation of cells, tissues, and organs. Theplacental mesoderm provides an ideal stromal environment, including anabundance of small molecules and growth factors, lipopolysaccharides,and extracellular matrix proteins, necessary for organogenesis andtissue neogenesis.

In one embodiment of the invention, the placenta can be populated withany particular cell type and used as a bioreactor for ex vivocultivation of cells, tissues or organs. Such cells, tissue or organcultures may be harvested used in transplantation and ex vivo treatmentprotocols. In this embodiment, the placenta is processed to remove allendogenous cells and to allow foreign (i.e., exogenous) cells to beintroduced and propagated in the environment of the perfused placenta.Methods for removal of the endogenous cells are well-known in the art.For example, the perfused placenta is irradiated with electromagnetic,UV, X-ray, gamma- or beta-radiation to eradicate all remaining viable,endogenous cells. The foreign cells of interest to be propagated in theirradiated placental bioreactor are then introduced, for example, byintroducing the cells via perfusion, via the vasculature or by directinjection into the placenta.

In another embodiment, the bioreactor may be used to produce andpropagate novel chimeric cells, tissues, or organs. Such chimeras may becreated using placental cells and one or more additional cell types asstarting materials in a bioreactor. The interaction, or “cross-talk”between the different cell types can induce expression patterns distinctfrom either of the starting cell types. In one embodiment, for example,an autologous chimera is generated by propagating a patient=s autologousplacental cells in a bioreactor with another cell type derived from thesame patient. In another embodiment, for example, a heterologous chimeramay be generated by addition of a patient=s cells, i.e., blood cells, toa bioreactor having heterologous placental cells. In yet anotherembodiment, the placental cells may be derived from a patient, and asecond cell type from a second patient. Chimeric cells are thenrecovered having a different phenotypic and/or genetic characteristicsfrom either of the starting cells. In a specific embodiment, theheterologous cells are of the same haplotype, and the chimeric cells arereintroduced into the patient.

In other embodiments, the bioreactor may be used for enhanced growth ofa particular cell type, whether native or synthetic in origin. Inanother embodiment of the invention, the placenta is used as abioreactor for propagating endogenous cells (i.e., cells that originatefrom the placenta), including but not limited to, various kinds ofpluripotent and/or totipotent embryonic-like stem cells and lymphocytes.In one embodiment, the placenta is incubated for varying periods of timewith perfusate solution as disclosed herein. Such endogenous cells ofplacental origin may be transformed to recombinantly express a gene ofinterest, to express mutations, and/or may be engineered to delete agenetic locus, using “knock out” technology. For example, an endogenoustarget gene may be deleted by inactivating or “knocking out” the targetgene or its promoter using targeted homologous recombination (e.g., seeSmithies, et al., 1985, Nature 317, 230-234; Thomas & Capecchi, 1987,Cell 51, 503-512; Thompson, et al., 1989, Cell 5, 313-321; each of whichis incorporated by reference herein in its entirety). For example, amutant, non-functional target gene (or a completely unrelated DNAsequence) flanked by DNA homologous to the endogenous target gene(either the coding regions or regulatory regions of the target gene) canbe used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express the target gene invivo. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the target gene. Suchapproaches may be used to remove, replace, or alter gene expression ofinterest in cells, tissue, and/or organs. This approach may be used toalter the phenotype of a cell, tissue, or organ, which may then beintroduced into a human subject.

In other embodiments, a placenta cell may be induced to differentiateinto a particular cell type, either ex vivo or in vivo. For example,pluripotent embryonic-like stem cells may be injected into a damagedorgan, and for organ neogenesis and repair of injury in vivo. Suchinjury may be due to such conditions and disorders including, but notlimited to, myocardial infarction, seizure disorder, multiple sclerosis,stroke, hypotension, cardiac arrest, ischemia, inflammation, age-relatedloss of cognitive function, radiation damage, cerebral palsy,neurodegenerative disease, Alzheimer's disease, Parkinsons's disease,Leigh disease, AIDS dementia, memory loss, amyotrophic lateralsclerosis, ischemic renal disease, brain or spinal cord trauma,heart-lung bypass, glaucoma, retinal ischemia, or retinal trauma.

The embryonic-like stem cells isolated from the placenta may be used, inspecific embodiments, in autologous or heterologous enzyme replacementtherapy to treat specific diseases or conditions, including, but notlimited to lysosomal storage diseases, such as Tay-Sachs, Niemann-Pick,Fabry's, Gaucher's, Hunter's, Hurler's syndromes, as well as othergangliosidoses, mucopolysaccharidoses, and glycogenoses.

In other embodiments, the cells may be used as autologous orheterologous transgene carriers in gene therapy to correct inborn errorsof metabolism or to treat cancer, tumors or other pathologicalconditions.

In other embodiments, the cells may be used in autologous orheterologous tissue regeneration or replacement therapies or protocols,including, but not limited to treatment of corneal epithelial defects,cartilage repair, facial dermabrasion, mucosal membranes, tympanicmembranes, intestinal linings, neurological structures (e.g., retina,auditory neurons in basilar membrane, olfactory neurons in olfactoryepithelium), burn and wound repair for traumatic injuries of the skin,or for reconstruction of other damaged or diseased organs or tissues.

The embryonic-like stem cells, progenitor cells, foreign cells, orengineered cells obtained from a placenta according to the methods ofthe invention can be used in the manufacture of a tissue or organ invivo. The methods of the invention encompass using cells obtained fromthe placenta, e.g., embryonic-like stem cells, progenitor cells, orforeign stem or progenitor cells, to seed a matrix and to be culturedunder the appropriate conditions to allow the cells to differentiate andpopulate the matrix. The tissues and organs obtained by the methods ofthe invention may be used for a variety of purposes, including researchand therapeutic purposes.

Uses of Embryonic-Like Stem Cells

The embryonic-like stem cells of the invention can be used for a widevariety of therapeutic protocols in which a tissue or organ of the bodyis augmented, repaired or replaced by the engraftment, transplantationor infusion of a desired cell population, such as a stem cell orprogenitor cell population. The embryonic-like stem cells of theinvention can be used to replace or augment existing tissues, tointroduce new or altered tissues, or to join together biological tissuesor structures.

For example, embryonic-like stem cells of the invention can be used intherapeutic transplantation protocols, e.g., to augment or replace stemor progenitor cells of organs or tissues such as the liver, pancreas,kidney, lung, nervous system, muscular system, bone, bone marrow,thymus, spleen, mucosal tissue, gonads, or hair.

Embryonic-like stem cells may be used instead of specific classes ofprogenitor cells (e.g., chondrocytes, hepatocytes, hematopoietic cells,pancreatic parenchymal cells, neuroblasts, muscle progenitor cells,etc.) in therapeutic or research protocols in which progenitor cellswould typically be used.

Embryonic-like stem cells of the invention can be used for augmentation,repair or replacement of cartilage, tendon, or ligaments. For example,in certain embodiments, prostheses (e.g., hip prostheses) are coatedwith replacement cartilage tissue constructs grown from embryonic-likestem cells of the invention. In other embodiments, joints (e.g., knee)are reconstructed with cartilage tissue constructs grown fromembryonic-like stem cells. Cartilage tissue constructs can also beemployed in major reconstructive surgery for different types of joints(for protocols, see e.g., Resnick, D., and Niwayama, G., eds., 1988,Diagnosis of Bone and Joint Disorders, 2d ed., W. B. Saunders Co.).

The embryonic-like stem cells of the invention can be used to repairdamage of tissues and organs resulting from disease. In such anembodiment, a patient can be administered embryonic-like stem cells toregenerate or restore tissues or organs which have been damaged as aconsequence of disease, e.g., enhance immune system followingchemotherapy or radiation, repair heart tissue following myocardialinfarction.

In other embodiments, the cells may be used in autologous orheterologous tissue regeneration or replacement therapies or protocols,including, but not limited to treatment of corneal epithelial defects,cartilage repair, facial dermabrasion, mucosal membranes, tympanicmembranes, intestinal linings, neurological structures (e.g., retina,auditory neurons in basilar membrane, olfactory neurons in olfactoryepithelium), burn and wound repair for traumatic injuries of the skin,scalp (hair) transplantation, or for reconstruction of other damaged ordiseased organs or tissues.

EXAMPLES Example 1 Analysis of Cell Types Recovered from Perfusate ofDrained Placenta

This example describes the analysis of the cell types recovered from theeffluent perfusate of a placenta cultured according to the methods ofthe invention.

Twenty ml of phosphate buffered saline solution (PBS) was added to theperfusion liquid and a 10 ml portion was collected and centrifuged for25 minutes at 3000 rpm (revolutions per minute). The effluent wasdivided into four tubes and placed in an ice bath. 2.5 ml of a 1% fetalcalf serum (FCS) solution in PBS was added and the tubes werecentrifuged (140 minutes×10 g (acceleration due to gravity)). The pelletwas resuspended in 5 ml of 1% FCS and two tubes were combined. The totalmononucleocytes were calculated by adding the total lymphocytes and thetotal monocytes, and then multiplying the result by the total cellsuspension volume.

The following table discloses the types of cells obtained by perfusionof a cultured placenta according to the methods described hereinabove.

WBC Total # of 1000/ml Lym % MID % GRA % Volume Cells CB 10.5 43.2 848.8 60 ml 6.3 × 10⁸ (Cord Blood) PP 12.0 62.9 18.2 18.9 15 ml 1.8 × 10⁸(Placenta perfusate, room temperature) PP₂ 11.7 56.0 19.2 24.8 30 ml 3.5× 10⁸ (Placenta perfusate, 37 degrees C.)

-   -   Samples of PP were after Ficoll.    -   Total cell number of PP after Ficoll was 5.3×10⁸ and number of        CB before processing is 6.3×10⁸. Lym % indicates percent of        lymphocytes; MID % indicates percent of midrange white blood        cells; and GRA % indicates percent of granulocytes.

Example 2 Analysis of Cells Obtained by Perfusion and Incubation ofPlacenta

The following example describes an analysis of cells obtained byperfusion and incubation of placenta according to the methods of theinvention.

Materials and Methods

Placenta donors were recruited from expectant mothers that enrolled inprivate umbilical cord blood banking programs and provided informedconsent permitting the use of the exsanguinated placenta followingrecovery of cord blood for research purposes. All donor data isconfidential. These donors also permitted use of blinded data generatedfrom the normal processing of their umbilical cord blood specimens forcryopreservation. This allowed comparison between the composition of thecollected cord blood and the effluent perfusate recovered using theexperimental method described below.

Following exsanguination of cord blood from the umbilical cord andplacenta, according to the methods described hereinabove, the placentawas placed in a sterile, insulated container at room temperature anddelivered to the laboratory within 4 hours of birth. Placentas werediscarded if, on inspection, they had evidence of physical damage suchas fragmentation of the organ or avulsion of umbilical vessels.Placentas were maintained at room temperature (23 plus/minus 2 degreesC.) or refrigerated (4 degrees C.) in sterile containers for 2 to 20hours. Periodically, the placentas were immersed and washed in sterilesaline at 25 degrees plus/minus 3 degrees C. to remove any visiblesurface blood or debris.

The umbilical cord was transected approximately 5 cm from its insertioninto the placenta and the umbilical vessels were cannulated with TEFLONor polypropylene catheters connected to a sterile fluid path allowingbi-directional perfusion of the placenta and recovery of the effluentfluid. The methods described hereinabove enabled all aspects ofplacental conditioning, perfusion and effluent collection to beperformed under controlled ambient atmospheric conditions as well asreal-time monitoring of intravascular pressure and flow rates, core andperfusate temperatures and recovered effluent volumes. A range ofconditioning protocols were evaluated over a 24-hour postpartum period,and the cellular composition of the effluent fluid was analyzed by flowcytometry, light microscopy and colony forming unit assays.

Placental Conditioning

The donor placentas were processed at room temperature within 12 to 24hours after delivery. Before processing, the membranes were removed andthe maternal site washed clean of residual blood. The umbilical vesselswere cannulated with catheters made from 20 gauge Butterfly needles usefor blood sample collection.

The donor placentas were maintained under varying conditions in anattempt to simulate and sustain a physiologically compatible environmentfor the proliferation and recruitment of residual embryonic-like stemcells. The cannula was flushed with IMDM serum-free medium (GibcoBRL,NY) containing 2 U/ml heparin (Elkins-Sinn, NJ). Perfusion of theplacenta continued at a rate of 50 ml per minute until approximately 150ml of perfusate was collected. This volume of perfusate was labeled“early fraction”. Continued perfusion of the placenta at the same rateresulted in the collection of a second fraction of approximately 150 mland was labeled “late fraction”. During the course of the procedure, theplacenta was gently massaged to aid in the perfusion process and assistin the recovery of cellular material. Effluent fluid was collected fromthe perfusion circuit by both gravity drainage and aspiration throughthe arterial cannula.

Placentas were then perfused with heparinized (2 U/ml) Dulbecco'smodified Eagle Medium (H.DMEM) at the rate of 15 ml/minute for 10minutes and the perfusates were collected from the maternal sites withinone hour and the nucleated cells counted. The perfusion and collectionprocedures were repeated once or twice until the number of recoverednucleated cells fell below 100/ml. The perfusates were pooled andsubjected to light centrifugation to remove platelets, debris andde-nucleated cell membranes. The nucleated cells were then isolated byFicoll-Hypaque density gradient centrifugation and after washing,resuspended in H.DMEM. For isolation of the adherent cells, aliquots of5-10×10⁶ cells were placed in each of several T-75 flasks and culturedwith commercially available Mesenchymal Stem Cell Growth Medium (MSCGM)obtained from BioWhittaker, and placed in a tissue culture incubator(37EC, 5% CO₂). After 10 to 15 days, the non-adherent cells were removedby washing with PBS, which was then replaced by MSCGM. The flasks wereexamined daily for the presence of various adherent cell types and inparticular, for identification and expansion of clusters offibroblastoid cells.

Cell Recovery and Isolation

Cells were recovered from the perfusates by centrifugation at X 00×g for15 minutes at room temperature. This procedure served to separate cellsfrom contaminating debris and platelets. The cell pellets wereresuspended in IMDM serum-free medium containing 2 U/ml heparin and 2 mMEDTA (GibcoBRL, NY). The total mononuclear cell fraction was isolatedusing Lymphoprep (Nycomed Pharma, Oslo, Norway) according to themanufacturer's recommended procedure and the mononuclear cell fractionwas resuspended. Cells were counted using a hemocytometer. Viability wasevaluated by trypan blue exclusion. Isolation of mesenchymal cells wasachieved by “differential trypsinization”, using a solution of 0.05%trypsin with 0.2% EDTA (Sigma, St. Louis Mo.). Differentialtrypsinization was possible because fibroblastoid cells detached fromplastic surfaces within about five minutes whereas the other adherentpopulations required more than 20-30 minutes incubation. The detachedfibroblastoid cells were harvested following trypsinization and trypsinneutralization, using Trypsin Neutralizing Solution (TNS, BioWhittaker).The cells were washed in H.DMEM and resuspended in MSCGM.

Flow cytometry was carried out using a Becton-Dickinson FACSCaliburinstrument and FITC and PE labeled monoclonal antibodies (mAbs),selected on the basis of known markers for bone marrow-derived MSC(mesenchymal stem cells), were purchased from B.D. and Caltaglaboratories (South San Francisco, Calif.), and SH2, SH3 and SH4antibody producing hybridomas were obtained from and reactivities of themAbs in their cultured supernatants were detected by FITC or PE labeledF(ab)′2 goat anti-mouse antibodies. Lineage differentiation was carriedout using commercially available induction and maintenance culture media(BioWhittaker), used as per manufacturer's instructions.

Isolation of Placental Embryonic-Like Stem Cells

Microscopic examination of the adherent cells in the culture flasksrevealed morphologically different cell types. Spindle-shaped cells,round cells with large nuclei and numerous perinuclear small vacuoles,and star-shaped cells with several projections (through one of whichstar-shaped cells were attached to the flask) were observed adhering tothe culture flasks. Although no attempts were made to furthercharacterize these adherent cells, similar cells were observed in theculture of bone marrow, cord and peripheral blood, and thereforeconsidered to be non-stem cell-like in nature. The fibroblastoid cells,appearing last as clusters, were candidates for being MSC (mesenchymalstem cells) and were isolated by differential trypsinization andsubcultured in secondary flasks. Phase microscopy of the rounded cells,after trypsinization, revealed that the cells were highly granulated;indistinguishable from the bone marrow-derived MSC produced in thelaboratory or purchased from BioWhittaker. When subcultured, theplacenta-derived embryonic-like stem cells, in contrast to their earlierphase, adhered within hours, assumed characteristic fibroblastoid shape,and formed a growth pattern identical to the reference bonemarrow-derived MSC. During subculturing and refeeding, moreover, theloosely bound mononuclear cells were washed out and the culturesremained homogeneous and devoid of any visible non-fibroblastoid cellcontaminants.

Results

The expression of CD-34, CD-38, and other stem cell-associated surfacemarkers on early and late fraction purified mononuclear cells wasassessed by flow cytometry. Recovered, sorted cells were washed in PBSand then double-stained with antiCD34 phycoerythrin and anti-CD38fluorescein isothiocyanate (Becton Dickinson, Mountain View, Calif.).

Cell isolation was achieved by using magnetic cell separation, such asfor example, Auto Macs (Miltenyi). Preferably, CD 34+ cell isolation isperformed first.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

The foregoing description of a preferred embodiment of the invention hasbeen presented for purposes of illustration and description. It is notintended to be exhaustive or to limit the invention precise formdisclosed. Obvious modifications or variations are possible in light ofthe above teachings. All such obvious modifications and variations areintended to be within the scope of the appended claims.

1.-23. (canceled)
 24. A tissue comprising a decellularized organ and aplurality of non-placental cells.
 25. The tissue of claim 24, whereinsaid decellularized organ is a placenta.
 26. The tissue of claim 24,wherein said decellularized organ is a heart.
 27. The tissue of claim24, wherein the non-placental cells are stem cells or progenitor cells.28. The tissue of claim 27, wherein the progenitor cells are progenitorcells for neurons, muscle, blood, bone, bone marrow, skin, heart,connective tissue, lung, kidney, liver, or pancreas.
 29. The tissue ofclaim 28, wherein said progenitor cells for pancreas are progenitorcells for pancreatic islet cells.
 30. The tissue of claim 27, whereinthe stem cells are mesenchymal stem cells.
 31. The tissue of claim 30,wherein said mesenchymal stem cells are from bone marrow.
 32. The tissueof claim 30, wherein said mesenchymal stem cells are from embryonic yolksac, umbilical cord, fetal skin, adolescent skin, or blood.
 33. Thetissue of claim 24, wherein the non-placental cells are on or more ofhematopoietic cells, neuron cells, fibroblast cells, strand cells, orhepatic cells.
 34. The tissue of claim 24, wherein the non-placentalcells are stromal cells, endothelial cells, hepatocytes, orkeratinocytes.
 35. The tissue of claim 24, wherein the non-placentalcells are cells that have been differentiated from placental stem cells.36. The tissue of claim 35, wherein the cells that have beendifferentiated from placental stem cells are adipogenic cells,chondrogenic cells, osteogenic cells, myogenic cells, vasogenic cells,neurogenic cells, or hepatogenic cells.